Methods for treating or preventing restenosis and other vascular proliferative disorders

ABSTRACT

Described herein is the use of ribonucleotide reductase inhibitors in the prevention or treatment of restenosis and other vascular proliferative disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. ProvisionalApplication No. 60/566,004, filed Apr. 28, 2004. U.S. ProvisionalApplication No. 60/566,004 is incorporated by reference herein in itsentirety.

The research leading to this invention was funded in part by NationalInstitute of Neurological Disorders and Stroke, Grant No. 1 R43NS044780-01A1 and the National Institutes of Health, Grants NS044780 andHL63744. The U.S. Government may have certain rights in this invention.

FIELD

Disclosed herein is the use of ribonucleotide reductase inhibitors inthe prevention or treatment of restenosis or other vascularproliferative disorders.

BACKGROUND

Restenosis is the narrowing of the blood vessels, which can occur afterinjury to the vessel wall, for example, injury caused by surgicaltechniques employed to achieve revascularization. Restenosis can occurfollowing a number of surgical procedures, for example, balloonangioplasty, transplant surgery, vein grafting, coronary by-passgrafting, and most frequently by percutaneous transluminal vascularintervention (PTVI). Unfortunately, many of these techniques are plaguedby a high incidence of vessel renarrowing or restenosis. Restenosis ismediated by internal hyperplasia and is characterized by excessiveproliferation of vascular smooth muscle cells in the walls of thetreated blood vessel.

The vascular changes observed following PTVI involve a cascade ofmolecular and cellular events occurring within the vessel wall involvingthe release of a variety of vasoactive, thrombogenic, and mitogenicfactors. These factors, acting in concert, stimulate the migration andproliferation of vascular smooth muscle cells (VSMC) and production ofextracellular matrix material, which accumulate to occlude the bloodvessel. Within this cascade are two processes that stand out aspotential therapeutic targets; vascular remodeling and neointimalhyperplasia. Vascular remodeling, defined as any enduring change in thesize and/or composition of a blood vessel, allows adaptation and repair.On the other hand, inappropriate remodeling, including its absence,underlies the pathogenesis of major cardiovascular diseases, such asatherosclerosis and restenosis. Experimental evidence acquired in vitroand in vivo suggests that the major drivers of vascular remodeling:inflammation, partially a result of oxidative stress and free radicalformation; and inappropriate cell migration and proliferation are allkey regulators in the remodeling process and ultimately lead to vesselrestenosis.

It has been demonstrated that cytokines can trigger much of theinflammatory component of injury. Cytokines are molecules that mediatethe migration of leukocytes into inflamed tissues and control theinflammatory reactions in various immune-mediated diseases. Both inanimal models and in human specimens, chemokine expression is associatedwith atherosclerotic lesion development and vascular remodeling andrestenosis after angioplasty. Activation of these inflammatory andprocoagulant mechanisms is thought to contribute significantly to theinitiation of restenosis. During this process, expression of tissuefactor (TF) represents another one of the major physiologic triggers ofcoagulation that results in thrombus formation and the generation ofadditional signals leading to VSMC proliferation and migration.

In addition, the activity of NF-kappa B, a transcriptional regulatoryfactor, has been demonstrated to be involved in the inflammatoryactivation of endothelial cells and their adhesiveness and also appearsto regulate apoptosis in smooth muscle cells (SMC) by coordinatinganti-apoptotic processes. The level of activity of NF-kappa B has beenshown to be enhanced within human atheromas or following angioplasty ascompared with healthy vessels. Thus, the inhibition of NF-kappa Bmobilization through therapeutic intervention can act to suppressendothelial activation and to induce SMC apoptosis. As mentioned, freeradicals and oxidant stress also play a role in vascular pathology.Increased oxidative stress is a major characteristic of restenosis afterangioplasty. The oxidative stress is mainly created by oxidants such asreactive oxygen species (ROS), which are assumed to play a role inneointima formation and smooth muscle proliferation. Once oxidant stressis invoked, characteristic pathophysiologic features follow, namelycompromised vessel reactivity, vascular smooth muscle cellproliferation, macrophage adhesion, platelet activation, and lipidperoxidation, all perpetrators in the restenosis process. Evidencesuggests that this complex cascade of molecular and cellular eventsoccurring within the vessel wall culminates in cellular proliferation.

What is needed is a method of preventing or treating restenosis andother vascular proliferative disorders. The compositions and methodsdisclosed herein meet this and other needs.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, and methods, as embodied and broadly described herein, thedisclosed subject matter, in one aspect, relates to compounds andcompositions and methods for preparing and using such compounds andcompositions. In another aspect, disclosed herein is the use ofribonucleotide reductase inhibitors for medical therapy in theprevention or treatment of restenosis.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

DESCRIPTION OF THE FIGURES

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and, togetherwith the description, illustrate the disclosed compositions and methods.

FIG. 1 is a series of photographs showing the effects of Didox, Imidate,and Hydroxyurea (HU) on the histopathology associated with balloondilatation injury. Examples are from carotid artery sections stainedwith hematoxylin and eosin. Panel (A) represents a control (uninjured)section of rat carotid artery. Panel (B) demonstrates the markedneointimal hyperplasia present at 2 weeks post balloon dilatationinjury. Panel (C) demonstrates the inhibitory effects of Didox (200 mgkg⁻¹ d⁻¹) on neointimal proliferation in response to balloon injury.Panel (D) demonstrates the inhibitory effects of Imidate (200 mg kg¹d⁻¹) on neointimal proliferation in response to balloon injury. Panel(E) demonstrates the inhibitory effects of HU (200 mg kg¹ d⁻¹) onneointimal proliferation in response to balloon injury. Note the markedreduction in neointimal (NI) thickness following Didox, Imidate, and HUtreatment.

FIGS. 2 A, B, and C are graphs showing the effects of Didox, Imidate,and HU on histopathological changes following balloon injury of the ratcarotid artery at 2 weeks post injury. Morphometric analysis wasperformed at the conclusion of the study. FIG. 2A compares neointimaformation (mm²). FIG. 2B compares medial wall thickness (mm²). FIG. 2Ccompares intima to media ratio. “Control” represents the uninjuredcontralateral artery. “Injured” represents the ipsilateral balloondilated artery. “Didox” represents groups that were administered Didoximmediately following injury followed by daily administration for 6days. “Imidate” represents groups that were administered Imidateimmediately following injury followed by daily administration for 6days. “HU” represents groups that were administered HU immediatelyfollowing injury followed by daily administration for 6 days. The datarepresent the mean±SD. “*” indicates significantly different at p<0.05as compared to injured (untreated). “‡” indicats significantly differentat p<0.05 among treated groups.

FIGS. 3 A, B, and C are graphs showing the effects of Didox, Imidate,and HU on histopathological changes following balloon injury of the ratcarotid artery at 6 weeks post injury. Morphometric analysis wasperformed at the conclusion of the study. FIG. 3A compares neointimaformation (mm²). FIG. 3B compares medial wall thickness (mm²). FIG. 3Ccompares intima to media ratio. “Control” represents the uninjuredcontralateral artery. “Injured” represents the ipsilateral balloondilated artery. “Didox” represents groups that were administered Didoximmediately following injury followed by daily administration for 6days. “Imidate” represents groups that were administered Imidateimmediately following injury followed by daily administration for 6days. “HU” represents groups that were administered HU immediatelyfollowing injury followed by daily administration for 6 days. The datarepresent the mean±SD. “*” indicates significantly different at p<0.05as compared to injured (untreated). “‡” indicats significantly differentat p<0.05 among treated groups.

FIG. 4 is a graph showing the effect of Didox, Imidate, and HU on SMCproliferation. The graph compares percent SMC growth inhibition as afunction of drug concentration (μM). Didox (0-200 μM), Imidate (0-200μM), and HU (0-1000 μM) were added to the SMC culture during the logphase of growth and incubated for 24 hours. Cells were then countedusing a flow cytometer. Values represent the mean. (n=4).

FIG. 5 is a graph showing the effects of Didox, Imidate, and HU on SMCmigration. The graph compares migration rate (μm/hr) as a function ofconcentration (μM). Didox (0-100 μM), Imidate (0-100 μM), and HU (0-1000μM) were added to the SMC culture in media containing 0.1% serum and 10ng/mL platelet derived growth factor (PDGF). A wound scrape was thenmade and SMC migration was monitored for an additional 24 hours. Thedata represent the mean±SD. “*” indicates significantly different atp<0.05 as compared to control.

DETAILED DESCRIPTION

The materials, compounds, compositions, articles, devices, and methodsdescribed herein can be understood more readily by reference to thefollowing detailed description of specific aspects of the disclosedsubject matter and the Examples included herein and to the Figures.

Before the present materials, compounds, compositions, components,and/or methods are disclosed and described, it is to be understood thatthe aspects described below are not limited to specific compounds,synthetic methods, or uses as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

GENERAL DEFINITIONS

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

Throughout the specification and claims the word “comprise” and otherforms of the word, such as “comprising” and “comprises,” means includingbut not limited to, and is not intended to exclude, for example, otheradditives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes mixtures of two or more such compounds, reference to “an agent”includes mixtures of two or more such agents, reference to “the moiety”includes mixtures of two or more such moieties, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed then “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe application, data are provided in a number of different formats, andthat this data, represent endpoints and starting points, and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point “15” are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15.

References in the specification and concluding claims to parts byweight, of a particular element or component in a composition orarticle, denotes the weight relationship between the element orcomponent and any other elements or components in the composition orarticle for which a part by weight is expressed. Thus, in a compoundcontaining 2 parts by weight of component X and 5 parts by weightcomponent Y, X and Y are present at a weight ratio of 2:5, and arepresent in such ratio regardless of whether additional components arecontained in the compound.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

By “inhibit” or other forms of the word such as “inhibiting” or“inhibition” is meant to hinder or restrain a particular characteristic.It is understood that this is typically in relation to some standard orexpected value, in other words it is relative, but that it is not alwaysnecessary for the standard or relative value to be referred to. Forexample, “inhibits ribonucleotide reductase” means hindering orrestraining the activity of the enzyme relative to a standard or acontrol. “Inhibits ribonucleotide reductase” can also mean to hinder orrestrain the synthesis or expression of the enzyme relative to astandard or control.

“Treatment” or “treating” means to administer a composition to a subjector a system with an undesired condition (e.g., restenosis or othervascular proliferative disorder). The condition can include a disease.“Prevention” or “preventing” means to administer a composition to asubject or a system at risk for the condition. The condition can includea predisposition to a disease. The effect of the administration of thecomposition to the subject (either treating and/or preventing) can be,but is not limited to, the cessation of a particular sysmptom of acondition, a reduction or prevention of the symptoms of a condition, areduction in the severity of the condition, the complete ablation of thecondition, a stabilization or delay of the development or progression ofa particular event or characteristic, or minimization of the chancesthat a particular event or characteristic will occur. It is understoodthat where treat or prevent are used, unless specifically indicatedotherwise, the use of the other word is also expressly disclosed.

By “subject” is meant an individual. The subject can be a mammal such asa primate or a human. The term “subject” can also include domesticatedanimals including, but not limited to, cats, dogs, etc., livestock(e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals(e.g., mouse, rabbit, rat, guinea pig, etc.).

Chemical Definitions

Disclosed herein are materials, compounds, compositions, and componentsthat can be used for, can be used in conjunction with, can be used inpreparation for, or are products of the disclosed methods andcompositions. These and other materials are disclosed herein, and it isunderstood that when combinations, subsets, interactions, groups, etc.of these materials are disclosed that while specific reference of eachvarious individual and collective combinations and permutation of thesecompounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a composition isdisclosed and a number of modifications that can be made to a number ofcomponents or moieties of the compositions are discussed, each and everycombination and permutation that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of components or moieties A, B, and C are disclosed as well as aclass of components or moieties D, E, and F, and an example of acombination composition A-D is disclosed, then even if each is notindividually recited, each is individually and collectivelycontemplated. Thus, in this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-group of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this disclosure including, but not limited to,steps in methods of making and using the disclosed compositions. Thus,if there are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific aspect or combination of aspects of the disclosed methods, andthat each such combination is specifically contemplated and should beconsidered disclosed.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. Also, as used herein “substitution” or “substitutedwith” is meant to encompass configurations where one substituent isfused to another substituent. For example, an alkyl group substitutedwith an aryl group can mean that the aryl group is bonded to the alkylgroup via a single sigma bond and also that the aryl group and alkylgroup are fused, e.g., two carbons of the alkyl group are shared withtwo carbons of the aryl group.

“A,” “A¹,” “A²,” “A³,” and “A⁴” are used herein as generic symbols torepresent various specific substituents. These symbols can be anysubstituent, not limited to those disclosed herein, and when they aredefined to be certain substituents in one instance, they can, in anotherinstance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 30 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl,tetracosyl, and the like. The alkyl group can also be substituted orunsubstituted. The alkyl group can be substituted with one or moregroups including, but not limited to, alkyl, halogenated alkyl, alkoxy,alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid,ester, ether, halide, hydroxy, ketone, sulfo-oxo, sulfonylamino, nitro,silyl, or thiol, as described below.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halides, e.g., fluorine, chlorine,bromine, or iodine. In another example, the term “alkylalcohol”specifically refers to an alkyl group that is substituted with one ormore hydroxyl groups, as described below, and the like. When “alkyl” isused in one instance and a specific term such as “alkylalcohol” is usedin another, it is not meant to imply that the term “alkyl” does not alsorefer to specific terms such as “alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “acyl” refers to both unsubstituted and substitutedacyl moieties, the substituted moieties can, in addition, bespecifically identified herein; for example, a particular substitutedacyl can be referred to as, e.g., an “acylamino.” Similarly, asubstituted alkoxy can be specifically referred to as, e.g., a“halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an“alkenylalcohol,” and the like. Again, the practice of using a generalterm, such as “acyl,” and a specific term, such as “acylamino,” is notmeant to imply that the general term does not also include the specificterm.

The term “alkoxy” as used herein is an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group may bedefined as —OA where A is alkyl as defined above.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 30carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This may be presumedin structural formulae herein wherein an asymmetric alkene is present,or it may be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, sulfo-oxo, sulfonylamino, nitro, silyl, or thiol, asdescribed below.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 30carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be substituted with oneor more groups including, but not limited to, alkyl, halogenated alkyl,alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylica cid, ester, ether, halide, hydroxy, ketone, sulfo-oxo, sulfonylamino,nitro, silyl, or thiol, as described below.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, sulfo-oxo, sulfonylamino, or thiol as described herein.The term “biaryl” is a specific type of aryl group and is included inthe definition of aryl. Biaryl refers to two aryl groups that are boundtogether via a fused ring structure, as in naphthalene, or are attachedvia one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group asdefined above where at least one of the carbon atoms of the ring issubstituted with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkylgroup can be substituted or unsubstituted. The cycloalkyl group andheterocycloalkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, sulfo-oxo, sulfonylamino, nitro, silyl, or thiol asdescribed herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and contains at least onedouble bound, e.g., C═C. Examples of cycloalkenyl groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term“heterocycloalkenyl” is a type of cycloalkenyl group as defined above,and is included within the meaning of the term “cycloalkenyl,” where atleast one of the carbon atoms of the ring is substituted with aheteroatom such as, but not limited to, nitrogen, oxygen, sulfur, orphosphorus. The cycloalkenyl group and heterocycloalkenyl group can besubstituted or unsubstituted. The cycloalkenyl group andheterocycloalkenyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, sulfo-oxo, sulfonylamino, nitro, silyl, or thiol asdescribed herein.

The term “cyclic group” is used herein to refer to either aryl groups,non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl groups), or both. Cyclic groups have one or more ringsystems that can be substituted or unsubstituted. A cyclic group cancontain one or more aryl groups, one or more non-aryl groups, or one ormore aryl groups and one or more non-aryl groups.

The term “acyl” as used herein is represented by the formula A-C═O,where A can be an alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,sulfo-oxo, sulfonylamino, nitro, silyl, or thiol as described herein.

The terms “amine” or “amino” as used herein are represented by theformula NA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen,an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl,cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl groupdescribed above.

The term “ester” as used herein is represented by the formula —OC(O)A or—C(O)OA, where A can be an alkyl, halogenated alkyl, alkenyl, alkynyl,aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, orheterocycloalkenyl group described above.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “halide” or “halo” as used herein refers to the halogensfluorine, chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen, alkyl, halogenatedalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group describedabove.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A (sulfoxide), —S(O)₂A (sulfonyl), —OS(O)₂A (sulfone), or—OS(O)₂OA, where A can be hydrogen, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “sulfonylamino” or “sulfonamide” as used herein is representedby the formula —S(O)₂NH—.

The term “thiol” as used herein is represented by the formula —SH.

“X,” “X^(n),” “Z,” “P,” “R,” and “R^(n),” where n is some integer, asused herein can, independently, possess two or more of the groups listedabove. These and other specific examples are disclosed herein. Forexample, if R is a straight chain alkyl group, one of the hydrogen atomsof the alkyl group can optionally be substituted with a hydroxyl group(OH), an alkoxy group, halide, etc. Depending upon the groups that areselected, a first group can be incorporated within second group or,alternatively, the first group can be pendant (i.e., attached) to thesecond group.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, components, and methods,examples of which are illustrated in the following description andexamples, and in the figures and their previous and followingdescription.

Ribonucleotide Reductase

Disclosed herein, in one aspect, are methods of treating or preventingrestenosis and other vascular proliferative disorders by administeringone or more ribonucleotide reductase inhibitors. Ribonucleotidereductase catalyzes the reductive conversion of ribonucleotides todeoxynucleotides. This reductive reaction is a rate limiting step in thebiochemical pathway leading to DNA synthesis and thus cell replication(Elford et al., (1970) J Biol Chem 245:5228-5233; Elford et al., (1979)Cancer Res 39:844-851; Takeda and Weber, (1981) Life Sci 28:1007-1014;Natsumeda et al., (1985) Cancer Res 45:2556-2559; Anderson et al.,(1993) J Interv Cardiol 6:187-202; Tanaka et al., (2000) Nature404:42-49). DNA synthesis cannot occur without invoking this reactionsince the endogenous pools of dNTP in mammalian cells are inadequate tosupport new DNA synthesis (Elford et al., (1970) J Biol Chem245:5228-5233; Elford et al., (1979) Cancer Res 39:844-851). Asdisclosed herein, ribonucleotide reductase is a prime target forimpeding cellular proliferation, and is amenable to inhibiting VSMCreplication and leukocyte production. It has been shown that rate ofuncontrolled cell growth is closely associated with the specificactivity of this enzyme. Because restenosis is ultimately aproliferative disease, ribonucleotide reductase inhibitors can serve asnovel therapeutic agents in the treatment of restenosis followingvascular trauma or injury. Also ribonucleotide reductase inhibitors canserve as novel therapeutic agents in the treatment of other disordercaused by abnormal vascular proliferation.

Ribonucleotide reductase comprises two subunits R1 and R2. The accessionnumbers for R1 and R2 subunits of homosapiens ribonucleotide reductaseare X59617 and X59618, respectively.

Ribonucleotide Reductase Inhibitors

The ribonucleotide reductase inhibitors disclosed herein can be used forthe treatment or prevention of restenosis or a vascular proliferativedisorder in a subject. By “ribonucleotide reductase inhibitor” is meantany composition that impairs or inhibits the enzyme ribonucleotidereductase or any of its subunits, which, as noted herein, catalyzes thereductive reaction that converts ribonucleotides intodeoxyribonucleotides. Such inhibitors can, for example, act by bindingto the enzyme, preventing the enzyme from becoming active, degrade theenzyme, alter expression of the enzyme, or alter the production of theenzymer.

The reductive reaction that converts ribonucleotides intodeoxyribonucleotides primarily occurs at the diphosphate form of thenucleotides. Suitable ribonucleotide reductase inhibitors can be smallmolecules that bind to or associate with the reductase, nucleic acidbased inhibitors that reduce or prevent the expression of ribonucleotidereductase, or polypeptide based molecules that interact with thereductase to inhibit enzyme activity. Also included are deoxynucleosidesthat are converted in vivo to deoxynucleotide di- or tri-phosphates thatinteract with RR holoenzyme or it subunits to interfere with theactivity of the enzyme. In one aspect, suitable examples ofribonucleotide reductase inhibitors include, but are not limited to,nucleoside inhibitors (e.g., fludarabine, 2-fluoromethylene-2′deoxycitidine-5′ diphoshate, gemcitibine, 2′ azido-2′ deoxynucleoside ofcytosine, and uracil thionucleotides), Fe-chelators (e.g., Desferrol,Desferoxamine, Desferri-Exochelin, and IC202C), thiosemicarbazides suchas Triapine, small peptide inhibitors such as P7, and hydroxamic acidssuch as hydroxyurea. These and other ribonucleotide reductase inhibitorsare described more fully herein.

Small Molecule Based Ribonucleotide Reductase Inhibitors

In one aspect, the ribonucleotide reductase inhibitors useful hereinhave the Formula I

wherein n is from 2 to 5, m is 0 or 1, R is NH₂, NHOH, OC₁₋₃ alkyl, orO-phenyl, R¹ is O, NH, NOH, or S and R² is H, OH, SH, or NH₂. It iscontemplated that Formula I includes the tautomeric form where the C═R¹moiety exists as a single bond (C—R¹), and the double bond exists in themoiety (CR²)_(m)═C. In this tautomeric form, the variables are the sameas disclosed above and further include R¹ being SH. In another aspect,prodrugs of the compounds having the Formula I can be used. For example,phenolic acetyl derivatives of compounds according to Formula I can beused as the ribonucleotide reductase inhibitors. In this aspect, theacetyl derivatives act as “pro-drugs” in that they are converted by thesubject to the corresponding ribonucleotide reductase inhibitor havingentirely unesterified phenolic hydroxyls, which are the therapeuticallyactive drugs.

Examples of ribonucleotide reductase inhibitors covered under Formula Iinclude, but are not limited to, 2,3-dihydroxyphenyl,3,4-dihydroxyphenyl, 2,5-dihydroxyphenyl, 2,6-dihydroxyphenyl,2,3,4-trihydroxyphenyl, 2,3,5-trihydroxyphenyl, 3,4,5-trihydroxyphenyl,2,4,5-trihydroxyphenyl, 2,3,4,5-tetrahydroxyphenyl, pentahydroxyphenyland the like groups.

In one aspect, when the ribonucleotide reductase inhibitor has theFormula I, m is 1 and R² is H (a phenylacetic acid derivative). Inanother aspect, when the ribonucleotide reductase inhibitor has theFormula I, m is 1 and R² is OH (a mandelic acid derivative). In afurther aspect, when the ribonucleotide reductase inhibitor has theFormula I, m is 0, R is NHOH and R¹ is O (an N-hydroxybenzamide(formerly, a benzohydroxamic acid)). In another aspect, when theribonucleotide reductase inhibitor has the Formula I, R is NH₂ and R¹ isNH (a benzimidamide (formerly a benzamidine)). In a further aspect, whenthe ribonucleotide reductase inhibitor has the Formula I, R is NHOH andR¹ is NH (an N-hydroxy benzimidamide (formerly a benzamidoxime)). Inanother aspect, when the ribonucleotide reductase inhibitor has theFormula I, R is NHOH and R¹ is NOH (an N,N′-dihydroxy benzimidamide(formerly, an hydroxyamidoxime)). In a further aspect, when theribonucleotide reductase inhibitor has the Formula I, R is O-alkyl orO-phenyl and R¹ is NH (a benzimidate). In another aspect, when theribonucleotide reductase inhibitor has the Formula I, R is OC₁₋₁₀ alkyl,wherein the alkyl groups include, but are not limited to, methyl, ethyl,isopropyl, and n-propyl.

Compounds represented by Formula I are disclosed in U.S. Pat. Nos.6,248,782; 4,253,322; 4,623,659; 2,848,430 and 3,629,443, which areincorporated by reference in their entireties. Methods for theirpreparation are also fully disclosed in these patents as well as in themany references cited therein. In another aspect, the polyphenoliccompounds N-3,4-trihydroxybenzamide (Didox) andethyl-3,4,5-trihydroxybenzenecarboximidate-HCl (Imidate) can be usedherein as ribonucleotide reductase inhibitors.

In another aspect, ribonucleotide reductase inhibitors having theFormula II can be used in the methods described herein

wherein n is from 2 to 5 and P can be COOH or thepharmaceutically-acceptable salt or ester thereof, CN, C₁₋₈ alkyl,aryl-substituted C₁₋₈ alkyl, acylamino, HOC₂H₄—NH—CH₂—C(═O)—,C₁-C₂H₄—NCH₃—CH₂—C(═O)—, C(S)OC₂H₅, C(O)—NH—C₁₋₃ alkyl,C(═NH)—N(OH)—C₁₋₃ alkyl, and substituted variants thereof. In oneaspect, phenolic blocking groups such as, for example, alkanoic acids,phenacyl esters and the like can be employed to yield pro-drugs whichare removed by the subject upon administration to yield drugs containingonly free phenolic hydroxyls.

In another aspect, ribonucleotide reductase inhibitors useful hereinhave the formula R³Z, wherein R³ can be H, NH₂, NH₂—NH, NHOH, NOH—R⁶,C₁₋₆ alkyl, OC₁₋₆ alkyl, aryl-substituted with C₁₋₆ alkyl, phenyl,naphthyl, pyridyl, pyrimidyl or thienyl, and wherein Z can beC(═O)NOH—R⁴, C(═S)—NOH—R⁴, C(═NH)—NOH—R⁴, C(═NOH)—C₁₋₃ alkyl, C(═NOH)—R⁴and C(═NOH)—R⁵, wherein R⁴ can be H, C₁₋₆ alkyl and substituted C₁₋₆alkyl, wherein R⁴ can be substituted with hydroxy, alkoxy, amino orhalo, and wherein R⁵ is NH₂ or NHOH, wherein R⁶ is C₁₋₆ acyl, alkyl andsubstituted C₁₋₆ alkyl substituted with hydroxyl, alkoxy, amino or haloand the like. The above group of compounds are generally referred to as“hydroxyureas.” The compounds disclosed in by Young, et al., (1967)Cancer Res. 27(Part 1):635, which is incorporated by reference in itsentirety, can be used herein. In one aspect, the ribonucleotidereductase inhibitors can be hydroxyurea.

In another aspect, ribonucleotide reductase inhibitors useful in themethods herein include semithiocarbazones such as, for example,2-formylpyridine, 2-acetylpyridine, 1-formylisoquinoline,1-acetylisoquinoline, and their ring substituted analogs. Formulae IIIand IV below illustrate these compounds

wherein Formula III represents pyridine-type compounds and Formula IVrepresents isoquinoline-semithiocarbazones, where X¹ can be H or CH₃, X²can be H, OH, NH₂, F, CF₃, C₁₋₃ alkyl, OX³, NHX³N(X³)₂, and O(O═C)X⁴, inwhich X³ denotes C₁₋₃ alkyl and X⁴ can be aryl, C₁₋₆ alkyl includingsubstitutions on the alkyl chain of the carboxylic acid with C₁₋₃alkoxy, C₁₋₃ mono- or di-alkylamino, aryloxy, also those in which thearyl ring is substituted with one or more hydroxy, amino or chlorogroups. Both E and Z isomers of the compounds and their mixtures can beused herein.

In one aspect, 2-formylpyridine and 2-acetylpyridine thiosemicarbazonederivatives useful herein include 3-hydroxy, 3-amino, 3-methyl,3-methoxy, 3-acetoxy, 3-ethoxy, 3-fluoro, 5-hydroxy, 5-amino, 5-fluoro,5-trifluoromethyl, 5-methoxy, 5-ethoxy, 5-dimethylamino, 5-pivaloyloxy,5-phenoxyacetoxy, 5-N,N-dimethylaminoacetoxy, and3,4-dihydroxybenzoyloxy as ring substituents. In another aspect,1-formylisoquinoline and 1-acetylisoquinoline thiosemicarbazonederivatives useful herein include 4-hydroxy, 4-methyl, 4-amino,5-fluoro, 5-trifluoromethyl, 5-amino and 5-acetylamino as ringsubstituents. The 2-formylpyridine and 1-formylisoquinolinethiosemicarbazone derivatives disclosed in French et al, (1974) J MedChem 17:172, which is incorporated by reference in its entirety, can beused herein. Other examples of thiosemicarbazone derivatives that can beused in the compositons and methods disclosed herein are described inU.S. Pat. Nos. 5,869,676, and 5,767,134, which are incorpored byreference herein for at least their teachings of thiosemicarbazonederivatives.

Still other examples of small molecule ribonucleotide reductaseinhibitors that can be used in the disclosed compositions and methodsare disclosed in U.S. Pat. Nos. 5,760,210, 5,569,666, 5,589,587,5,550,144, 5,545,650, 5,545,649, 5,541,207, 5,536,739, 5,536,738,5,519,041, 5,514,695, 5,508,393, 5,498,622, 5,496,841, 5,496,840,5,496,839, 5,496,838, 5,496,837, 5,476,841, 5,466,702, 5,391,563,5,292,775, 5,198,425, 5,173,505, 5,128,353, 5,071,835, 4,845,195,4,837,304, 4,814,432, which are incorporated by reference herein for atleast their teachings of ribonucleotide reductase inhibitors.

Nucleic Acid Based Inhibitors

In another aspect, a suitable ribonucleotide reductase inhibitor can bea nucleic acid based inhibitor. There are a variety of moleculesdisclosed herein that are nucleic acid based. The disclosed nucleicacids are made up of for example, nucleotides, nucleotide analogs, ornucleotide substitutes. Non-limiting examples of these and othermolecules are discussed herein. It is understood that for example, whena vector is expressed in a cell, that the expressed mRNA will typicallybe made up of A, C, G, and U. Likewise, it is understood that if, forexample, an antisense molecule is introduced into a cell or cellenvironment through for example exogenous delivery, it is advantageousthat the antisense molecule be made up of nucleotide analogs that reducethe degradation of the antisense molecule in the cellular environment.

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenine-9-yl (A),cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymine-1-yl(T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc Natl AcadSci USA (1989) 86:6553-6556).

Antisense

Antisense molecules are one example of suitable nucleic acid basedribonucleotide reductase inhibitors that can be used in the disclosedmethods and compositions. Antisense molecules are designed to interactwith a target nucleic acid molecule through either canonical ornon-canonical base pairing. The interaction of the antisense moleculeand the target molecule (e.g., ribonucleotide reductase or a subunitthereof) is designed to promote the destruction of the target moleculethrough, for example, RNAseH mediated RNA-DNA hybrid degradation.Alternatively the antisense molecule is designed to interrupt aprocessing function that normally would take place on the targetmolecule, such as transcription or replication. Antisense molecules canbe designed based on the sequence of the target molecule. Numerousmethods for optimization of antisense efficiency by finding the mostaccessible regions of the target molecule exist. Exemplary methods wouldbe in vitro selection experiments and DNA modification studies using DMSand DEPC. It is preferred that antisense molecules bind the targetmolecule with a dissociation constant (k_(d)) less than or equal to10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². A representative sample of methods andtechniques which aid in the design and use of antisense molecules can befound in the following non-limiting list of U.S. Pat. Nos. 5,135,917,5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138,5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320,5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042,6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

To design antisense oligonucleotides, a host mRNA sequence is examined.Regions of the sequence containing multiple repeats, such as TTTTTTTT,are not as desirable because they will lack specificity. Severaldifferent regions can be chosen. Of those, oligonucleotides are selectedby the following characteristics: those having the best conformation insolution; those optimized for hybridization characteristics; and thosehaving less potential to form secondary structures. Antisense moleculeshaving a propensity to generate secondary structures are less desirable.

Suitable antisense molecules for the disclosed methods and compositionsimpair ribonucleotide reductase synthesis or activity. Antisensemolecules that can be used in the disclosed methods and compositions canalso be obtained commercially. For example, Lorus Therapeutics (Toronto,Canada) has developed antisense molecules which are specific for eitherR1 or R2 mRNA, the two subunits of ribonucleotide reductase. Specificexamples of antisense molecules from Lorus that can be used in thedisclosed methods and compositions include GTI-2040 and GTI-2501. Recenttoxicology studies with rodents and monkeys have indicated that thiscompound is likely to be safe in humans at concentrations that exceedtherapeutic doses. GTI-2040 (having following sequence5′-GGCTAAATCGCTCCACCAAG-3′ (SEQ ID NO:1)) is currently in a Phase IIclinical trial for patients with advanced or metastatic renal cellcarcinoma. GTI-2501 is in Phase II clinical trials for use inhormone-refractory prostate cancer in combination with docetaxel. Otherexamples of suitable antisense molecules that can be used in thedisclosed methods and compositions are disclosed in U.S. Pat. Nos.6,593,305, 6,121,000, and 5,998,383, which are incorporated by referenceherein in for at least their teachings of antisense ribonucleotidereductase inhibitors.

Short Interfering RNAs

Short interfering RNAs (siRNAs) are another example of suitable nucleicacid based ribonucleotide reductase inhibitors that can be used in thedisclosed methods and compositions. siRNAs are double-stranded RNAs thatcan induce sequence-specific post-transcriptional gene silencing,thereby decreasing or even inhibiting gene expression.

In one example, an siRNA triggers the specific degradation of homologousRNA molecules, such as mRNAs, within the region of sequence identitybetween both the siRNA and the target RNA. For example, WO 02/44321discloses siRNAs capable of sequence-specific degradation of targetmRNAs when base-paired with 3′ overhanging ends. The direction of dsRNAprocessing determines whether a sense or an antisense target RNA can becleaved by the produced siRNA endonuclease complex. Thus, siRNAs can beused to modulate transcription, for example, by silencing genes, such asB-chimerin rho-GTPase or Rab9. The effects of siRNAs have beendemonstrated in cells from a variety of organisms, including Drosophila,C. elegans, insects, frogs, plants, fungi, mice and humans (for example,WO 02/44321; Gitlin et al., (2002) Nature 418:430-4; Caplen et al.,(2001) Proc. Natl. Acad. Sci. 98:9742-9747; and Elbashir et al., (2001)Nature 411:494-8). In certain examples, siRNAs are directed againstcertain target genes, such as ribonucleotide reductase.

Examples of siRNA that can be used to inhibit the expression ofribonucleotide reductase, as disclosed herein, are disclosed in Duxburyet al., (2004) Oncogene. 23(8):1539-1548; Duxbury et al., (2004)Surgery. 136(2):261-269; Lin et al., (2004) Biol. Chem.,279(26):27030-27038; which are incorporated by reference herein for atleast their teachings of siRNA.

Polypeptide Based Ribonucleotide Reductase Inhibitors

In yet another aspect, a suitable ribonucleotide reductase inhibor canbe a polypeptide (for example, an antibody specific for ribonucleotidereductase or subunits R1, R2, or p53 thereof). Examples of polypeptidebased ribonucleotide reductase inhibitors are disclosed in U.S. Pat.Nos. 6,030,942 and 5,885,830, which are incorporated by reference hereinfor at least their teachings of polypeptide based ribonucleotideinhibitors. In one example, P7 is a suitable peptide basedribonucleotide reductase inhibitor having the sequence TLDADF (SEQ IDNO:2). P7 corresponds to the C-terminus of mR2 and competes with mR2 forbinding to mR1.

Pharmaceutically Acceptable Salts

Where possible, any of the compounds described herein can be thepharmaceutically acceptable salt or ester thereof. In one aspect,pharmaceutically acceptable salts are prepared by treating the free acidwith an appropriate amount of a pharmaceutically acceptable base.Representative pharmaceutically acceptable bases are ammonium hydroxide,sodium hydroxide, potassium hydroxide, lithium hydroxide, calciumhydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide,copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine, and the like. In one aspect, the reaction isconducted in water, alone or in combination with an inert,water-miscible organic solvent, at a temperature of from about 0° C. toabout 100° C. such as at room temperature. The molar ratio ofribonucleotide reductase inhibitor to base used is chosen to provide theratio desired for any particular salts. For preparing, for example, theammonium salts of the free acid starting material, the starting materialcan be treated with approximately one equivalent of pharmaceuticallyacceptable base to yield a neutral salt.

Ester derivatives are typically prepared as precursors to the acid formof the compounds and accordingly can serve as prodrugs. Generally, thesederivatives will be lower alkyl esters such as methyl, ethyl, and thelike. Amide derivatives —(CO)NH₂, —(CO)NHR and —(CO)NR₂, where R is analkyl group defined above, can be prepared by reaction of the carboxylicacid-containing compound with ammonia or a substituted amine.

Some of the ribonucleotide reductase inhibitors may potentially beadministered as a pharmaceutically acceptable acid- or base-additionsalt, formed by reaction with inorganic acids such as hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, and phosphoric acid, and organic acids such as formicacid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvicacid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaricacid, or by reaction with an inorganic base such as sodium hydroxide,ammonium hydroxide, potassium hydroxide, and organic bases such asmono-, di-, trialkyl and aryl amines and substituted ethanolamines. Inanother aspect, the ribonucleotide reductase inhibitor is in the form ofthe sodium or potassium salt. In another embodiment, the ribonucleotidereductase inhibitors can be converted to the correspondingpharmaceutically-acceptable ester such as, for example, the methylester.

Methods of Use

The disclosed ribonucleotide reductase inhibitors have a wide variety ofuses, for example, they can be used to treat or prevent restenosis orother vascular proliferative disorders following injury or varioussurgical procedures.

As an example, the disclosed ribonucleotide reductase inhibitors can beused to treat or prevent restenosis and other vascular proliferationdisorders in a subject in need thereof. In one aspect, a subject can beidentified as being at risk for restenosis or other vascularproliferation disorders. Subjects at risk for restenosis are generallythose who have undergon or are undergoing a vascular trauma, forexample, following percutaneous transluminal coronary angioplasty(PTCA). The use of PTCA has greatly reduced the number of fatalities inpatients who suffer myocardial infarction (Fischman et al., (1994) NEngl J Med 331:496-501; Elezi et al., (1998) Circulation 98:1875-1880;Bennett and O'Sullivan, (2001) Pharmacol Ther 91:149-166). During PTCA,the artery walls are expanded by several times their original diameterin an attempt to increase lumen diameter and improve flow.Unfortunately, this technique is plagued by a high incidence of vesselrenarrowing or restenosis occurring in 30-40% of patients within 6months of the procedure (Anderson et al., (1993) J Interv Cardiol6:187-202; Fischman et al., (1994) N Engl J Med 331:496-501; Elezi etal., (1998) Circulation 98:1875-1880; Bennett and O'Sullivan, (2001)Pharmacol Ther 91:149-166; Heckenkamp et al., (2002) J Cardiovasc Surg(Torino) 43:349-357). Prevention of restenosis after successful PTCAremains one of the most challenging tasks in the treatment ofobstructive coronary artery disease. Attempts to ameliorate thisproliferative response involve the use coronary stents, which havesignificantly improved both short term and long term outcome followinginterventional coronary revascularization procedures. Despite areduction in restenosis rate with stent deployment, restenosis stilloccurs in 15-30% of patients within 6 months (Fischman et al., (1994) NEngl J Med 331:496-501; Elezi et al., (1998) Circulation 98:1875-1880).This incidence of in-stent restenosis is expected to increase ascoronary stenting is becoming more frequent and is used in less ideallesions. Therefore, in addition to mechanical intervention,pharmacological approaches to reduce the incidence and degree ofrestenosis, such as those disclosed herein, are needed.

The vascular trauma associated with PTCA involves a cascade of molecularand cellular events occurring within the vessel wall involving therelease of a variety of vasoactive, thrombogenic, and mitogenic factors(Bauters and Isner, (1997) Prog Cardiovasc Dis 40:107-116; Libby andTanaka, (1997) Prog Cardiovasc Dis 40:97-106; Goldschmidt-Clermont andMoldovan, (1999) Gene Expr 7:255-260). Within this cascade, severalmechanisms contribute to restenosis including elastic recoil,thrombosis, smooth muscle cell migration/proliferation and matrixformation. The result of these vascular events is intimal hyperplasia,whereby vascular smooth muscle cells (VSMC's) migrate from the media tothe intima, proliferate, and consequently form the neointima. Duringthis proliferative response, SMCs undergo a phenotypic modulation from acontractile to a synthetic phenotype (differentiation) (Epstein et al.,(1991) Circulation 84:778-787; Noda-Heiny and Sobel, (1995) Am J Physiol268:C1195-1201; Ueda et al., (1995) Coron Artery Dis 6:71-81; Farb etal., (2002) Circulation 105:2974-2980; Indolfi et al., (2003) TrendsCardiovasc Med 13:142-148). While not wishing to be bound by theory, thecompounds and compositions are believed to be effective because theytarget/impede smooth muscle cell migration and cell proliferation,critical components of restenosis injury. Thus, the discloseribonucleotide reductase inhibitors can be used to treat or preventrestenosis and other vascular proliferative disorders.

i. Delivery

As used throughout, administration of any of the ribonucleotidereductase inhibitors described herein can occur in conjunction withother therapeutic agents. Thus, the ribonucleotide reductase inhibitorscan be administered alone or in combination with one or more therapeuticagents. For example, a subject can be treated with a ribonucleotidereductase inhibitor alone, or in combination with chemotherapeuticagents, antibodies, antibiotics, antivirals, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokines,chemokines and/or growth factors. Combinations can be administeredeither concomitantly (e.g., as an admixture), separately butsimultaneously (e.g., via separate intravenous lines into the samesubject), or sequentially (e.g., one of the compounds or agents is givenfirst followed by the second). Thus, the term “combination” or“combined” is used to refer to either concomitant, simultaneous, orsequential administration of two or more agents. In one aspect, theribonucleotide reductase inhibitors can be combined with other agentssuch as, for example, Paclitaxel, Taxotere, other taxoid compounds,other anti proliferative agents such as Methotrexate, anthracyclinessuch as doxorubicin, immunosuppressive agents such as Everolimus andSerolimus, and other rapamycin and rapamycin derivatives.

The ribonucleotide reductase inhibitors can be administered in a numberof ways depending on whether local or systemic treatment is desired, andon the area to be treated. Administration can be topically (includingopthamalically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedcompounds can be administered intravenously, intraarterialy,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, intratracheal, extracorporeally, or topically (e.g.,topical intranasal administration or administration by inhalant). Asused herein, “topical intranasal administration” means delivery of thecompositions into the nose and nasal passages through one or both of thenares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the ribonucleotide inhibitor ormendicant. The latter can be effective when a large number of subjectsare to be treated simultaneously. Administration of the compositions byinhalant can be through the nose or mouth via delivery by a spraying ordroplet mechanism. Delivery can also be directly to any area of therespiratory system (e.g., lungs) via intubation.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein in its entiretyfor the methods taught.

The compositions can be in solution or in suspension (for example,incorporated into microparticles, liposomes, or cells). Thesecompositions can be targeted to a particular cell type via antibodies,receptors, or receptor ligands. The following references are examples ofthe use of this technology to target specific proteins to given tissue(Senter et al., (1991) Bioconjugate Chem., 2:447-451; Bagshawe, (1989)Br. J. Cancer, 60:275-281; Bagshawe et al., (1988) Br. J. Cancer,58:700-703; Senter et al., (1993) Bioconjugate Chem., 4:3-9; Battelli etal., (1992) Cancer Immunol. Immunother. 35:421-425; Pietersz andMcKenzie, (1992) Immunolog. Reviews, 129:57-80; Roffler et al., (1991)Biochem. Pharmacol., 42:2062-2065). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. In general, receptors are involved in pathways of endocytosis,either constitutive or ligand induced. These receptors cluster inclathrin-coated pits, enter the cell via clathrin-coated vesicles, passthrough an acidified endosome in which the receptors are sorted, andthen either recycle to the cell surface, become stored intracellularly,or are degraded in lysosomes. The internalization pathways serve avariety of functions, such as nutrient uptake, removal of activatedproteins, clearance of macromolecules, opportunistic entry of virusesand toxins, dissociation and degradation of ligand, and receptor-levelregulation. Many receptors follow more than one intracellular pathway,depending on the cell type, receptor concentration, type of ligand,ligand valency, and ligand concentration. Molecular and cellularmechanisms of receptor-mediated endocytosis has been reviewed (Brown andGreene, (1991) DNA and Cell Biology 10:6, 399-409).

When nucleic acid based ribonucleotide inhibitors are used in thedisclosed methods, these compositions can be administrated and taken upinto the cells of a subject (i.e., gene transduction or transfection) inthe form of naked DNA or RNA, or the nucleic acids can be in a vectorfor delivering the nucleic acids to the cells, as would be wellunderstood by one of ordinary skill in the art. The vector can be acommercially available preparation, such as an adenovirus vector(Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of thenucleic acid or vector to cells can be via a variety of mechanisms. Asone example, delivery can be via a liposome, using commerciallyavailable liposome preparations such as LIPOFECTIN, LIPOFECTAMINE(GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden,Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as wellas other liposomes developed according to procedures standard in theart. In addition, the disclosed nucleic acid or vector can be deliveredin vivo by electroporation, the technology for which is available fromGenetronics, Inc. (San Diego, Calif.) as well as by means of aSONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., (1988) Proc. Natl. Acad. Sci. U.S.A.85:4486; Miller et al., (1986) Mol. Cell. Biol. 6:2895). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof). The exact method of introducing the alterednucleic acid into mammalian cells is, of course, not limited to the useof retroviral vectors. Other techniques are widely available for thisprocedure including the use of adenoviral vectors (Mitani et al., (1994)Hum. Gene Ther. 5:941-948), adeno-associated viral (AAV) vectors(Goodman et al., (1994) Blood 84:1492-1500), lentiviral vectors (Naidiniet al., (1996) Science 272:263-267), pseudotyped retroviral vectors(Agrawal et al., (1996) Exper. Hematol. 24:738-747). Physicaltransduction techniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms (see, for example,Schwartzenberger et al., (1996) Blood 87:472-478). This disclosedcompositions and methods can be used in conjunction with any of these orother commonly used gene transfer methods.

Parenteral administration of the nucleic acid or vector, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution of suspension in liquid prior to injection,or as emulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. For additionaldiscussion of suitable formulations and various routes of administrationof therapeutic compounds, see, e.g., Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995.

Amounts

As disclosed herein, the ribonucleotide reductase inhibitors areadministered to a subject in an effective amount. By “effective amount”is meant a therapeutic amount needed to achieve the desired result orresults, e.g., treating or preventing restenosis or other vascularproliferation disorders. The exact amount of the compositions requiredwill vary from subject to subject, depending on the species, age, weightand general condition of the subject, the severity of the disorder beingtreated, its mode of administration and the like. Thus, it is notpossible to specify an exact amount for every composition. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein.

ii. Pharmaceutically Acceptable Carriers

The ribonucleotide reductase inhibitors can be used therapeutically incombination with a pharmaceutically acceptable carrier. Pharmaceuticalcarriers are known to those skilled in the art. These most typicallywould be standard carriers for administration of drugs to humans,including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

In one aspect, any of the ribonucleotide reductase inhibitors describedherein can be combined with at least one pharmaceutically-acceptablecarrier to produce a pharmaceutical composition. The pharmaceuticalcompositions can be prepared using techniques known in the art. In oneaspect, the composition is prepared by admixing the ribonucleotidereductase inhibitor having with a pharmaceutically-acceptable carrier.The term “admixing” is defined as mixing the two components together sothat there is no chemical reaction or physical interaction. The term“admixing” also includes the chemical reaction or physical interactionbetween the ribonucleotide reductase inhibitor and thepharmaceutically-acceptable carrier.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

iii. Therapeutic Uses

In one aspect, disclosed are methods for treating or preventingrestenosis and other vascular proliferation disorders in a subject,comprising administering to the subject an effective amount of aribonucleotide reductase inhibitor. In one aspect, ribonucleotidereductase inhibitors can be used for the treatment of restenosis injuryfollowing a number of surgical techniques including, but not limited to,transplant surgery, vein grafting, coronary by-pass grafting, PTCA, andangioplasty.

In other aspects, ribonucleotide reductase inhibitors can be used forthe treatment of benign vascular proliferative disorders. Examples ofsuch disorders include, but are not limited to, vascular proliferationinvolved in atherosclerosis, vascular proliferation followingintravascular device implantation, vascular proliferation at the site ofvascular anastamosis as generally occurs following revascularizationprocedure or A-V shunting, vascular proliferation following carotidendarderectomy, and transplant vasculopathy.

Not wishing to be bound by theory, it is believed that ribonucleotidereductase inhibitors possess chemical and biological properties that canimpair several processes that contribute to the restenosis process orother vascular proliferative disorders. For example, members of thepolyphenolic series such as 3,4-dihydroxy- and3,4,5-trihydroxy-compounds have been shown to be good anti-inflammatoryagents by inhibiting NF-kappa B activation and, therefore, downregulating cytokines contributing to the inflammation process andchemoattractant production. Also these compounds can inhibit tissuefactor production. This synergistic concept can be a vasoprotectiveapproach to the prevention of restenosis or other vascular proliferationdisorders by attenuating inflammatory reactions, SMC proliferation, andneointima formation following vascular injury.

In one aspect, polyhydroxy phenolic compounds described herein have theability to modulate several of the biological events contributing to thedevelopment of restenosis, uncontrolled SMC proliferation, leukocytemigration, NF-kappa B activation and tissue factor expression, leadingto inflammation. These compounds being excellent free-radicalscavengers, are able to modulate the above mentioned deleteriousprocesses. Having these properties make these compounds unique to limitthe restenosis process or other vascular proliferation disorders.

In one aspect, a medical device can comprise one or more ribonucleotidereductase inhibitors in order to treat or prevent restenosis or othervascular proliferation disorders. In one aspect, an implant of a medicaldevice such as, for example, a stent, can contain the ribonucleotidereductase inhibitor. In other examples, the medical device can be aneedle, cannula, catheter, shunt, balloon, or valve. The ribonucleotidereductase inhibitor can be formulated to permit its incorporation ontothe medical device, which can apply the ribonucleotide reductaseinhibitor directly to the site to prevent or treat such restenosis orother vascular proliferation disorder. In one aspect, the ribonucleotidereductase inhibitor can be formulated by including it within a coatingon the medical device. There are various coatings that can be utilizedsuch as, for example, polymer coatings that can release theribonucleotide reductase inhibitor over a prescribed time period. Inanother aspect, the ribonucleotide reductase inhibitor can be embeddeddirectly into the medical device.

In another aspect, the ribonucleotide reductase inhibitors can play afurther positive role in preventing restenosis in organ transplantationor other surgical procedures where the incidence of viral infection ispossible. Examples of viral infections include, but are not limited to,Herpes viruses, particularly cytomegalovirus, herpes I and II, hepatitisB and C, as well as HIV I and II. Viral infections can contribute to therestenosis process caused by the surgical vascular procedures listedabove. Therefore, in this aspect, the ribonucleotide reductaseinhibitors can prevent restenosis by inhibiting viruses such as herpesand HIV replication and the possible inflammatory aspect of hepatitis Cthat contribute to restenosis.

EXAMPLES

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

The following examples test the ability of three ribonucleotidereductase inhibitors to limit the degree of restenosis following balloonmediated dilatation injury in the rat. Specifically, the ability of thepolyhydroxy-phenolic compounds, Didox (3,4-dihydroxybenzohydraxamicacid), Imidate (3,4,5-hydroxybenzimidate), and hydroxyurea have beenexamined to limit the degree of restenosis following vascular injury.

Example 1

To determine the ability of the ribonucleotide reductase inhibitors toaffect restenosis, a rat model of balloon mediated carotid artery injurywas utilized. Percutaneous transluminal carotid angioplasty (PTCA) wasperformed using a 2F Fogarty Embolectomy Catheter, which was insertedthrough the right, external carotid artery and inflated (2 atm) anddeflated four times inside the right common carotid artery and rotatedto cause endothelial injury. Immediately following the carotid injury,the rats received ribonucleotide reductase inhibitors, Didox(N,3,4-trihydroxybenzamide) Imidate(ethyl-3,4,5-trihydroxybenzenecarboximidate.HCl), or Hydroxyurea viaintraperitoneal injection. The degree of restenosis was assessed usingmorphometric analysis at two weeks post injury. For example,ribonucleotide reductase inhibition with Didox resulted in a 19%increase in lumen area, 84% decrease in neointimal formation, 48%reduction in total wall thickness and an 85% reduction in theintima/media ratio as compared to the untreated injured artery (Table1). These results suggest that ribonucleotide reductase does serve as anovel therapeutic target in the treatment of post PTCA restenosis.

TABLE 1 Effects of Ribonucleotide Reductase Inhibitors onRestenosis-Data presented as % change as compared to injured vessel.Wall Neointima/ Group Lumen Neointima Thickness Media Didox ↑ 19 ± 3% ↓84 ± 2% ↓ 48 ± 3% ↓ 85 ± 4% Imidate ↑ 19 ± 5% ↓ 65 ± 7% ↓ 41 ± 7% ↓ 69 ±8% Hydroxyurea ↑ 22 ± 5% ↓ 91 ± 6% ↓ 54 ± 9% ↓ 93 ± 7%

Example 2

Didox, Imidate, and Hydroxyurea were provided by Molecules for HealthInc. (Richmond, Va.). Rat vascular smooth muscle cells and culture mediawere purchased from ATCC (Manassas, Va.). Fogarty embolectomy catheterswere purchased from M & I medical (Miami, Fla.). All other reagents werepurchased from Sigma Chemical Co. (St. Louis, Mo.).

Carotid Injury

Male Wistar rats, weighing 400-450 g (Harlan) were fed standard pelletfeed and given water ad libitum. The experimental protocol was designedin accordance with Institutional ILACUC standards. Animals wereanesthetized with Isoflurane (1.5-2%) in air. The right carotid arterywas exposed and a 2F Fogarty balloon embolectomy catheter (Baxter) wasinserted via an external carotid arteriotomy incision. The catheter wasadvanced to the common carotid artery, inflated to a pressure of 2 atmand rotated in a forward and retrograde direction. The catheter was thendeflated and the process repeated three times. Treatment cohorts weredivided into 5 groups (n=6-8/group): Control (sham operated), Didox (200mg·kg⁻¹ d⁻¹), Imidate (200 mg kg⁻¹ d⁻¹), HU (200 mg kg⁻¹ d⁻¹) andvehicle (saline). Drugs were administered daily by i.p. injection for aperiod of 7 days after injury. At 2 weeks post-injury, rats wereeuthanized by pentobarbital overdose and perfused with 10% bufferedformalin. Carotid arteries were removed and placed in the same fixative.Tissues were then embedded in paraffin, and 4 to 5 sections (4 μm) werecut at multiple levels. These sections were then stained withhematoxylin-eosin or elastic-van Gieson stain. Sections were examinedmicroscopically and the cross-sectional areas of the lumen, neointima(from the internal elastic lamina to the lumen), and media weredetermined using digital microscopy with Spot Advanced software. Theintima-to-media (I/M) ratio was then calculated from the determinedmean. The data represent the mean±SD.

Flow Cytometry

SMC were plated on 6 well dishes at a density of 2×10⁵ cells per well.Cells were then treated with Didox (0-200 μM), Imidate (0-200 μM) or HU(0-1000 μM) and incubated for 24 hours. Following the 24 hour incubationperiod, the cells were trypsinized and collected in 15 mL centrifugetubes. The cells were then centrifuged 5 minutes at 800×g. Thesupernatant was discarded and the pellet was resuspended in 5 ml PBS.The cells were centrifuged 6 minutes at 200×g. The supernatant wasremoved and the pellet was then thoroughly resuspended in 0.5 ml PBS.The cell suspension was transferred into tubes containing 70% ethanol,keeping the cells in fixative for greater than about 2 hours. Theethanol suspended cells were centrifuged 5 minutes at 200×g, and theethanol was decanted. The cell pellet was resuspended in 5 ml PBS andafter one minute was centrifuged 5 min at 200×g. The top layer of liquidwas again removed and the cell pellet was resuspended in 1 ml PI/TritonX-100 staining solution with RNAse A. This staining solution was thenincubated at room temperature for 30 minutes. Flow cytometry was thenperformed using a FACS Calibur (Becton Dickenson). The data arepresented as the mean.

Intracellular dNTP Quantitation

SMC were plated in T150 flasks and treated with Didox (0-200 μM),Imidate (0-200 μM) or HU (0-1000 μM) and incubated for 24 hours.Following the 24 hour incubation period, the cells were trypsinized andcollected in 50 mL centrifuge tubes along with the incubation media. Allthe extraction steps were performed on ice. Immediately beforeprocessing, cells were counted and viability determined using the trypanblue exclusion method. The cells were then centrifuged 5 minutes at800×g. The cell pellet was then deproteinized with the same volume of 6%TCA, vortexed for 20s and incubated on ice for 10 minutes. The acid cellextracts were centrifuged 10 min at 2000×g. The supernatants were thensupplemented with an equal volume of distilled water, vortexed for 60 sand neutralized by the addition of 5M K₂CO₃ prior to HPLC analysis. dNTPdetection was carried out using an ESA (Chelmsford, Mass.) HPLCchromatographic system with UV-Vis detection. Chromatographicseparations were performed using a TosoHaas C18 reverse phase column(ODS 80Tm 250×4.6 mm, 5 μm pore). The mobile phase was delivered at arate of 1.0 mL/min during the analysis using the following stepwisegradient elution program: A-B (80-20) at 0 min; (40-60) at 30 min;(40-60) at 40 min and (80-20) at 45 min. Buffer A contained 10 mMtetrabutylammonium sulfate, 10 mM KH₂PO₄ and 0.25% MeOH, and adjusted topH 6.9. Buffer B consisted of 5.6 mM tetrabutylammonium sulfate, 50 mMKH₂PO₄ and 30% MeOH, and neutralized to pH 7.0. The injection volume foranalysis was 50 μL. Detection was carried out at 254 nm.

Smooth Muscle Cell Migration

A wound scrape assay was performed using rat vascular SMC. The cellswere grown to confluence on 60 mm dishes. The cells were then madequiescent by incubating them in media containing 0.1% serum for 24 hrsfollowed by treatment with Didox, Imidate (0-100 μM), or HU (0-1000 μM)in media containing 0.1% serum and platelet derived growth factor (PDGF)at concentration of 10 ng/mL. Linear wounds were made by scraping eachplate with the tip of a 20 μL pipette. The ability of cells to migrateacross the wound area was measured using digital microscopy. SPOTadvanced software (Diagnostic Instruments, Inc., Sterling Heights,Mich.) was used to measure the wound immediately, 2, 4, 12 and 24 hrsfollowing injury. The data are presented as rate of migration andrepresent the mean±SD.

Results: Effects of Ribonucleotide Reductase Inhibitors on RestenosisFollowing Vascular Injury

The effects of Didox, Imidate, and Hydroxyurea (HU) on the vascularremodeling process following arterial injury were determined using a ratmodel of balloon mediated carotid injury. Each compound (200 mg kg⁻¹d⁻¹) was delivered via i.p. injection for a period of 7 days postinjury. These dosages are based on previously published reports andrepresent ½ the maximum tolerated dose of Hydroyurea in rats (Gupta andYaffe, (1982) Pediatr Pharmacol (New York) 2:85-91; Vaughan et al.,(1989) Cancer Chemother Pharmacol 23:26-30). Furthermore, it haspreviously been demonstrated that these doses are sufficient to inhibitribonucleotide reductase activity without causing significant toxicityin mice and rats (Mayhew et al., (2002) Antivir Chem Chemother13:305-314; Mayhew et al., (1999) Stem Cells 17:345-356; Horvath et al.,(2004) Cancer Chemother Pharmacol. 54(2):139-145).

At 14 days post injury the animals were sacrificed and morphometricanalysis was carried out in order to asses the histopathological changesin the vessel wall (FIG. 1). Didox treatment resulted in a 62% decreasein neointimal area and a 61% decrease in intima/media ratio (FIG. 2).Imidate treated groups demonstrated a 57% decrease in neointimal areaand a 55% decrease in the intima/media ratio (FIG. 2). Because Didox andImidate possess other chemical attributes in addition to their effectson ribonucleotide reductase (Fritzer-Szekeres et al., (2000) Exp Hematol28:924-930; Fritzer-Szekeres et al., (1997) Life Sci 61:2231-2237;Fritzer-Szekeres et al., (2002) Biochem Pharmacol 64:481-485; Lee etal., (1997) Virology 234:277-290; Inayat et al., (2002) Cancer Biol Ther1:539-545) the commercially available ribonucleotide reductaseinhibitor, HU (200 mg kg⁻¹ d⁻¹), was also tested. HU afforded similarvascular protective effects to those observed with Didox and Imidate,resulting in a 55% decrease in neointimal area and a 63% decrease inintima/media ratio (FIG. 2). These results indicate that ribonucleotidereducatase inhibition can modulate the remodeling process followingvascular injury. However, because the remodeling process occurs over aprolonged period of time, additional studies were performed in order todetermine whether the vascular protective effects observed would bemitigated over time.

To further investigate long-term efficacy of the one week dosingregimen, the duration of the study period was increased to 6 weeks. Itwas found that the degree of neointimal thickening at 6 weekspost-injury was increased by 57% as compared to the 2 week studyparadigm (FIG. 3). The beneficial effect of ribonucleotide reductaseinhibition persisted over the long term. Each compound (200 mg kg⁻¹ d⁻¹)was given i.p. for a period of 7 days followed by a 5 week recoveryperiod. At the end of the 6 week period, the Didox treated groupexhibited a 64% decrease in neointimal area and a 71% decrease inintima/media ratio (FIG. 3). Similarly, Imidate offered a 61% reductionin neointimal area and a 62% reduction in intima/media ratio (FIG. 3).HU treatment reduced neointimal formation by 71% and decreased theintima/media ratio by 75% (FIG. 3). These results suggest thatactivation of ribonucleotide reductase is an early trigger in thevascular response to injury and that inhibition of this enzyme can limitthe neointimal proliferation associated with restenosis.

Effects of Didox, Imidate, and HU on SMC Proliferation.

Didox, Imidate, and to a much lesser degree, HU, are known to beribonucleotide reductase inhibitors (Elford, (1968) Biochem Biophys ResCommun 33:129-135; Elford et al., (1970) J Biol Chem 245:5228-5233;Elford et al., (1979) Cancer Res 39:844-851). ribonucleotide reductasecatalyzes the reductive conversion of ribonucleotides todeoxynucleotides. This reductive reaction is a prime target for impedingcellular proliferation, and therefore amenable to inhibiting VSMCreplication, because it is a rate limiting step in the biochemicalpathway leading to DNA synthesis (Elford et al., (1970) J Biol Chem245:5228-5233; Elford et al., (1979) Cancer Res 39:844-851; Takeda andWeber, (1981) Life Sci 28:1007-1014; Natsumeda et al., (1985) Cancer Res45:2556-2559). The ability of Didox and Imidate to inhibitribonucleotide reductase activity has been documented with publishedreports demonstrating an IC₅₀ of 3-30 μM for this class of compounds(Elford et al., (1979) Cancer Res 39:844-851). These values represent agreater than 10 fold increased effectiveness over the classicalribonucleotide reductase inhibitor HU (Elford, (1968) Biochem BiophysRes Commun 33:129-135). Based on this evidence, experiments wereperformed in order to determine whether the ability of these compoundsto reduce neointimal formation was due to their ability to inhibit SMCproliferation in-vitro. Therefore, the IC₅₀ of each compound oninhibition of smooth muscle cell growth was determined. Cells wereincubated in the presence of Didox (0-200 μM), Imidate (0-200 μM), andHU (0-1000 μM) for 24 hours. Cell numbers were then counted using flowcytometry and the concentration at which cell division was inhibited by50% (IC₅₀) was calculated. Didox yielded an IC₅₀ of 67 μM, Imidate wasslightly less potent exhibiting an IC₅₀ of 82 μM, while HU was the leastpotent with an IC₅₀ of 266 μM (FIG. 4). These results are consistentwith ribonucleotide reductase activity data and demonstrate that Didoxand Imidate are 3-4 times more potent than HU at arresting cell divisionin vascular smooth muscle cells.

In order to further validate that the observed anti-proliferativeproperties afforded by these compounds was through inhibition ofribonucleotide reductase activity, the effects of these compounds onintracellular dATP pools were measured. Results demonstrated that Didox(0-200 μM), Imidate (0-200 μM), and HU (0-1000 μM) dose dependentlydepleted the endogenous dNTP pools with maximal reductions in dATPcontent of 58%, 42% and 69%, respectively (Table 2).

TABLE 2 Effects of Didox, Imidate and HU on SMC dATP pools. Didox (0-200μM), Imidate (0-200 μM) and HU (0-1000 μM) were added to the SMC cultureduring the log phase of growth and incubated for 24 hours. dNTP's wereextracted and samples subjected to HPLC analysis. The data are presentedas pmoles dATP/107 cells and represent the mean ±SD. “*” indicatessignificantly different at p < 0.05 as compared to control. HydroxyureaControl Didox (μM) Imidate (μM) (μM) Dose 0 5 10 50 100 200 5 10 50 100200 5 100 500 1000 dATP 303 297 260 203* 140* 126* 293 311 210 184* 173*320 266 93 92 levels (Pmoles/ 10⁷ cells) S.D. 50 35 26 20 40 31 31 17 4741 39 30 42 11 17

The concentrations at which these drugs afford their in-vitro biologicaleffects are well below the range of the peak plasma levels (300-400 μM)measured following Didox and Imidate infusion (200 mg kg⁻¹ d⁻¹) andwould be expected to inhibit ribonucleotide reductase activity based onthe published and observed K_(i)'s for these compounds (Elford et al.,(1979) Cancer Res 39:844-851). These results indicate that part of thevascular protective effects of these compounds is due to their abilityto impede SMC proliferation.

Effects of Didox, Imidate, and HU on SMC Migration.

SMC migration is also a component of neointimal proliferation.Therefore, additional studies were performed in order to determine theeffects of these compounds on SMC migration. Using a wound scrape assay,SMC migration studies were carried out in the presence of Didox,Imidate, and HU. VSMC's were cultured to confluence on 60 mm dishes. Thecells were made quiescent by incubating in media containing 0.1% serum.Following 24 hours of serum deprivation, Didox, Imidate, and HU wereadded to the wells (10-1000 μM) in the presence of PDGF (10 ng/mL) and alinear wound was made across the plate. SMC migration across the woundwas monitored by digital microscopy over a 24 hour period. Results fromthese studies demonstrated that Didox (100 μM) and Imidate (100 μM)treatment almost completely inhibited SMC migration, decreasing themigratory rate from 15.8 μM/hr in the control to 1.7 μM/hr and 0.9μM/hr, respectively (FIG. 5). In contrast, HLT (100 μM) had littleeffect on SMC migration, resulting in a migratory rate of 15.1 μM/hr(FIG. 4). No further inhibition was seen with HU concentrations up to 1mM. These results demonstrate that Didox and Imidate significantlyimpair SMC migration. This would be expected to contribute to thevascular protective effects afforded by these drugs. However,ribonucleotide reductase inhibition appears to be the principalmechanism as the rat arterial injury data demonstrated similar efficacywith HU.

Discussion

Although PTCA and coronary artery stenting have had a tremendous impacton the treatment of coronary vascular disease, these procedures aremarked by a high incidence of restenosis (Anderson et al., (1993) JInterv Cardiol 6:187-202; Fischman et al., (1994) N Engl J Med331:496-501; Elezi et al., (1998) Circulation 98:1875-1880; Bennett andO'Sullivan, (2001) Pharmacol Ther 91:149-166; Heckenkamp et al., (2002)J Cardiovasc Surg (Torino) 43:349-357). This process of vesselre-narrowing is characterized by neointimal hyperplasia resulting inlumen loss and impaired vascular function. The vascular response toinjury triggers a migratory and proliferative response within the smoothmuscle cells resulting in intimal thickening (Noda-Heiny and Sobel,(1995) Am J Physiol 268:C1195-1201; Libby and Tanaka, (1997) ProgCardiovasc Dis 40:97-106; Elezi et al., (1998) Circulation 98:1875-1880;Ward et al., (2000) Circulation 102:1186-1191; Bennett and O'Sullivan,(2001) Pharmacol Ther 91:149-166; Heckenkamp et al., (2002) J CardiovascSurg (Torino) 43:349-357; Lanza et al., (2002) Circulation106:2842-2847; Segev et al., (2002) Cardiovasc Res 53:232-241; Bhargavaet al., (2003) Bmj 327:274-279; Crook and Akyurek (2003) TrendsCardiovasc Med 13:102-106; Indolfi et al., (2003) Trends Cardiovasc Med13:142-148). In this regard, emphasis has been placed on developingpharmacological therapy aimed at reducing the proliferative response.Currently, two pharmacological agents have been approved for clinicaluse in the treatment of post-PTCA restenosis (Drachman et al., (2000) JAm Coll Cardiol 36:2325-2332; Heldman et al., (2001) Circulation103:2289-2295; Suzuki et al., (2001) Circulation 104:1188-1193). Taxoland Rapamyacin are being delivered through the use of coated coronarystents. Preliminary results suggest that these approaches offersignificant protection against the restenosis process and validate theuse of antiproliferative agents in the treatment of vascularproliferative disorders such as restenosis (Sousa et al., (2001)Circulation 103:192-195; Gershlick et al., (2004) Circulation109:487-493; Stone et al., (2004) Circulation 109:1942-1947).

The use of ribonucleotide reductase inhibition has been demonstratedherein as a new therapeutic target in ameliorating balloon mediatedrestenosis injury. The biochemical attributes of this enzyme make itamenable for the treatment of proliferative disorders since inhibitionof ribonucleotide reductase blocks DNA synthesis and thus cellreplication. These results herein demonstrate that following ballooninjury, one week of systemic administration of the ribonucleotidereductase inhibitors, Didox and Imidate, largely inhibited neointimalformation resulting in a 60% reduction in the intima/media ratio.Morphometric analysis revealed an about 60% reduction in neointimal areawith no significant change in the medial area between treated anduntreated groups. However, there was a small but statisticallysignificant decrease in medial area following Imidate dosing when thiscohort was compared against HU and Didox. These results indicate thatImidate can have some negative effects on smooth muscle remodelingfollowing medial injury that result in medial wall thinning.

These disclosed in-vivo studies demonstrate that inhibition ofribonucleotide reductase limits the extent of intimal hyperplasiafollowing mechanical injury. Also, these compounds possess a variety ofchemical attributes that can contribute to their protective effects.Didox has been shown to inhibit NF-kappa B and Tissue Factor expression.In addition both Didox and Imidate compounds are potent free radicalscavengers (Fritzer-Szekeres et al., (1997) Life Sci 61:2231-2237; Leeet al., (1997) Virology 234:277-290; Duilio et al., (2001) Am J PhysiolHeart Circ Physiol 280:H2649-2657; Shet et al., (2003) Blood102:2678-2683; Turchan et al., (2003) Neurology 60:307-314; InayatCancer Biol Ther. (2002) 1(5):539-545). Because of the myriad of effectselicited by these compounds, further experiments were carried out usingthe commercially available ribonucleotide reductase inhibitor, HU.Following the same dosing regimen for Didox and Imidate, HU affordedsimilar anti-restenotic efficacy, further supporting the observationdescribed herein regarding the importance of ribonucleotide reductase inthe vascular response to injury.

Because the vascular response to injury is a chronic process, additionalstudies were performed in order to assess whether the protective effectselicited by early ribonucleotide reductase inhibition are maintainedthroughout the remodeling period. The ribonucleotide reductaseinhibitors (Didox, Imidate, and HU) were administered daily for one weekand the extent of injury assessed at 6 weeks after balloon dilatation.This additional recovery time resulted in a greater than 50% increase inthe intima/media ratio demonstrating the progression of the lesion overtime. Interestingly, Didox and HU treatment reduced the intima/mediaratio by greater than 70%, while slightly less efficacy was observedwith Imidate. Moreover, the degree of protection afforded by thesecompounds was significantly increased when compared to the results ofthe two week study. This suggests that ribonucleotide reductase is anearly target in the vascular response to injury and that inhibition ofthis enzyme affects the long term vascular remodeling associated withrestenosis.

These results demonstrate that ribonucleotide reductase inhibitionlimits the degree of restenosis following arterial dilation injury.While not wishing to be bound by theory, it is believed that theseeffects are mediated through an inhibition of SMC proliferation, as thisprocess precedes neointimal formation. Therefore, studies were performedusing a cell proliferation assay. Didox, Imidate, and HU treatmentresulted in arrest of cell division. Analysis of dNTP pools demonstrateda greater than 50% reduction in dATP levels further supporting that theobserved effects of these drugs are mediated at least in part throughthe inhibition of ribonucleotide reductase. These effects wereindependent of any cytotoxic action these compounds may posses as flowcytometry revealed less than 3% apoptotic cells following the dosingregimes tested. In addition, prior to dNTP analysis, cells were countedand viability assessed using trypan blue exclusion. Cell viability wasgreater than 95% among all groups.

As previously stated, Didox and Imidate possess various chemicalattributes in addition to ribonucleotide reductase inhibition, many ofwhich can confer protection against restenosis (Fritzer-Szekeres et al.,(1997) Life Sci 61:2231-2237; Lee et al., (1997) Virology 234:277-290;Duilio et al., (2001) Am J Physiol Heart Circ Physiol 280:H2649-2657;Shet et al., (2003) Blood 102:2678-2683; Turchan et al., (2003)Neurology 60:307-314). Among these is their ability to scavenge freeradicals, which can modulate SMC migration, a component of neointimalproliferation (Duilio et al., (2001) Am J Physiol Heart Circ Plzysiol280:H2649-265; Turchan et al., (2003) Neurology 60:307-314). Therefore,the effects of Didox and Imidate were tested on SMC proliferation usinga wound scrape assay. Results demonstrated that both compounds almostcompletely inhibited SMC migration. This indicates that inhibition ofSMC proliferation can contribute to the protection afforded by thesecompounds. Similarly, a number of studies have demonstrated that HU,when oxidized, can release NO. (Stolze and Nohl, (1990) BiochemPharmacol 40:799-802; Pacelli et al., (1996) Lancet 347:900). Because NOhas been shown to inhibit cell migration, it was tested whether HUpossesses anti-migratory properties which could be involved in itsanti-restenotic effects (Sarkar et al., (1997) Am J Physiol272:H1810-1818; Sarkar and Webb, (1998) J Vasc Res 35:135-142). HU atconcentrations up to 1 mM had no effect on SMC migration. Thisobservation indicates that inhibition of ribonucleotide reductase is theprincipal mechanism through which these compounds afford theirprotection as similar efficacy was seen with all compounds. However, ifthe in-vivo results are interpreted on a molar basis, HU (2.6 mmoleskg⁻¹ d⁻¹) doses are 2-3 fold higher than that of Didox (1.2 mmoles kg¹d⁻¹) and Imidate (0.9 mmoles kg¹ d⁻¹) and suggests that the effects ofDidox and Imidate on SMC migration can contribute to the vascularprotective effects observed with these two compounds.

Taken together, the in-vivo and in-vitro data demonstrate thatactivation of ribonucleotide reductase is an early component in theproliferative response associated with vascular injury and thatinhibition of this enzyme can reduce the vascular pathology associatedwith restenosis injury. Although the incidence of restenosis hasmarkedly decreased with the advent of drug coated stents, restenosisstill occurs in up to 20% of patients within the first year whileresults on late lumen loss are still being gathered (Drachman et al.,(2000) J Am Coll Cardiol 36:2325-2332; Heldman et al., (2001)Circulation 103:2289-2295; Grube et al., (2003) Circulation 107:38-42;Kastrati et al., (2005) JAMA 293:165-171; Kim et al., (2005) Heart91:e15). In addition, because the use of coated stents may increase therisk of thrombosis there is a need for agents which can be administeredsystemically with or without stents in patients at high risk forthrombotic events (Morice, (2005) JACC 45). Inhibition of ribonucleotidereductase can be a pathway that can be therapeutically targeted througheither local or systemic delivery based on the low toxicity associatedwith current ribonucleotide reductase inhibitor therapy using HU.Additionally, the data disclosed herein suggest that ribonucleotidereductase is an early target in the restenosis process and as such,early pharmacological intervention can preclude chronic therapy and itsassociated adverse side effects. It is believed that these observationshave therapeutic potential and implicate ribonucleotide reductase as apromising therapeutic target in the treatment of vascular proliferativedisorders.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

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1. A method of treating or preventing restenosis comprisingadministering to a subject who has restenosis, or a subject who isundergoing or has undergone a vascular procedure, in an amount effectiveto treat restenosis or prevent restenosis due to the vascular procedure,a composition that inhibits ribonucleotide reductase, wherein thecomposition comprises N-3,4-trihydroxybenzamide or a pharmaceuticallyacceptable salt or ester thereof.
 2. The method of claim 1, wherein thesubject is undergoing or has undergone a vascular procedure.
 3. Themethod of claim 2, wherein the vascular procedure comprises balloonangioplasty.
 4. The method of claim 2, wherein the vascular procedurecomprises transplant surgery.
 5. The method of claim 2, wherein thevascular procedure comprises a vein graft.
 6. The method of claim 2,wherein the vascular procedure comprises a coronary by-pass graft. 7.The method of claim 2, wherein, the vascular procedure comprises apercutaneous transluminal vascular intervention (PTVI).
 8. The method ofclaim 2, wherein the vascular procedure comprises intravascular deviceimplantation.
 9. The method of claim 2, wherein the vascular procedurecomprises carotid endarderectomy.
 10. The method of claim 1, furthercomprising determining the degree of restenosis after administering thecomposition.